2.4. Primary pancreatic islet isolation

Pancreatic islets were isolated using collagenase type V digestion as previously described (Cawthorn & Chan, 1991; Zhang et al., 2001). Briefly, 12‐week‐old C57BL/6J mice were anaesthetized, and their pancreatic ducts were cannulated and injected with 1 mg/ml type V collagenase (Sigma‐Aldrich, St. Louis, MO, USA) in D‐Hank's buffered saline solution to inflate the pancreases. The pancreases were then carefully isolated and loaded into digestion solution containing 1 mg/ml type V collagenase. After digestion at 37°C for approximately 28 min, the islets were dispersed via manual agitation. The digested tissue was resuspended in 5 ml of Histopaque (Sigma‐Aldrich, St. Louis, MO, USA) that was then overlaid with 5 ml of D‐Hank's solution. After gradient centrifugation, the islets were collected and transferred to RPMI 1640 medium supplemented with 10% FBS. The islets were cultured at 37°C in a humidified atmosphere consisting of 5% CO₂/95% air for 12 h (primary culture) to remove exocrine and other tissues. A total of 1200 islets isolated from 10–12 mice were cultured and treated with or without FFAs. After 24 h, pancreatic islets and their conditioned media were collected for RNA analysis or exosome isolation.